In vitro maturation and fertilization of bovine oocytes and in vitro culture of presumptive zygotes in the presence of bovine pestivirus

Abstract

A pathogen which has been shown to commonly contaminate in vitro bovine embryo production system is bovine pestivirus (bovine viral diarrhea virus). Three experiments were designed to evaluate the in vitro maturation (experiment I), fertilization (experiment II) and embryo development (experiment III) of immature oocytes, inseminated oocytes and presumptive zygotes in the presence of a bovine pestivirus (non-cytopathic, nCP type 1). The virus inoculum used was derived from a persistently infected cow. In experiment I, follicular oocytes ( n=1257) recovered from slaughterhouse derived ovaries were randomly assigned to either a control group ( n=578) which did not become exposed to bovine pestivirus and a treatment group ( n=679) which was inoculated with bovine pestivirus (2.20–3.69 log 10 TCID 50/50 μl) at the time of commencement of in vitro maturation. Overall, there was no significant difference between the control and pestivirus inoculated oocytes in either the cumulus cell expansion rate (79±7.5% versus 74±10.7%) or the nuclear maturation rate (89±4.8% versus 85±7.4%), respectively. In experiment II, in vitro matured oocytes ( n=607) were inseminated either in the absence (control; n=301) or the presence of bovine pestivirus (4–4.6 log 10 TCID 50/50 μl; n=306). A significant ( P<0.01) reduction in the overall number of fertilized oocytes with two well formed male and female pronuclei was observed in the treatment group compared to the control group (58.5±5.8% versus 73.3±3.6%, respectively). In experiment III, after in vitro maturation and fertilization, presumptive zygotes were randomly assigned to either a control group ( n=139) which was not exposed to bovine pestivirus or a treatment group which was inoculated with bovine pestivirus (2.97–4.47 log 10 TCID 50/30 μl; n=139). The zygotes were then cultured under mineral oil in an atmosphere of 88% N 2, 7% O 2 and 5% CO 2 at 39 °C. The morphologic appearance of the embryos was assessed 48 h after the commencement of culture, and then every 48 h up to days 7–8 after insemination. The 22% (31/139) and 3.6% (5/139) of the presumptive zygotes developed to the morula or blastocyst stage in the control and the bovine pestivirus inoculated groups, respectively ( P<0.001). This study demonstrates that bovine pestivirus has a significant detrimental effect on in vitro fertilization and early in vitro embryo development.